This application addresses broad Challenge Area #8, Genomics, and specific Challenge Topic 08-HL-101, which seeks to identify causal genetic variants associated with heart, lung, and blood diseases by application of targeted DNA capture and massively parallel sequencing technologies followed by selective genotyping of DNA samples from large well-phenotyped populations. We are particularly focused on element (b), disease or clinical-trait based exome-wide resequencing for the unbiased discovery of rare variants having large effects. Severe combined immunodeficiency (SCID) is a syndrome characterized by absent T and B lymphocyte function that is uniformly fatal in infancy without successful immune reconstitution. SCID patients typically lack T cells but have varying numbers of B cells and natural killer (NK) cells depending on the molecular defect. To date, mutations in 15 different genes have been shown to cause SCID. Most of the molecular etiologies of SCID were delineated by analysis of candidate genes that encode components of lymphocyte cytokine receptors, structural components of T cell receptor complex, products required for VDJ recombination, or proteins important in lymphocyte survival or function. However, a recent report combining genome-wide SNP analysis with functional screening of candidates in zebrafish surprisingly found that mutations in the mitochondrial energy metabolism gene adenylate kinase 2 (AK2) were responsible for the SCID variant reticular dysgenesis in 6 individuals. This report demonstrates the feasibility of our planned genome-wide analyses to uncover SCID-causing mutations in novel genes that would not have been elucidated by standard approaches. Of the 202 SCID patients evaluated at Duke University, the molecular etiology remains uncertain in 29 individuals despite examination for known causes of this syndrome. This patient population represents the largest cohort of SCID patients in the United States and sufficient numbers of frozen cells are available from all of these patients for the studies proposed in this application. The vast majority of gene mutations shown to cause SCID have two characteristics: 1) they result in bi-allelic inactivation;and 2) they occur in the coding regions or adjacent canonical intron splice sites of the genes in question. Accordingly, we propose to identify the causal mutations in the SCID patients of unknown etiology by sequencing the exomes of those patients (along with those of unaffected siblings or parents), bioinformatically identifying candidate genes that are bi-alellically inactivated and likely to be responsible for disease, and then functionally screening those candidate genes from patients representing 3 patient classes in zebrafish and murine models. These models are ideal for this purpose as essential elements of hematopoiesis are conserved and they enable rapid screening of effects of gene inactivation on development using either shRNAs or morpholinos. We feel that the advent of deep sequencing methodologies affords us an opportunity to make great gains scientifically as well as to accomplish the goals of the stimulus package by both supporting the efforts of biotech companies and providing high-tech jobs. PUBLIC HEALTH RELEVANCE: Our strategy for identifying novel causes of SCID promises to provide great and potentially paradigm-shifting insights in both biology and medicine. Indeed, identification of the genes causing SCID in our cohort of 29 patients is crucial for: 1) better understanding of immune system development and function;2) genetic counseling and prenatal diagnosis;and 3) possible future application of gene therapy to the treatment of these infants.